Formalin

Fixation with formalin is a standard method for conserving tissues for histological and
immunohistochemical analysis because it yields superior morphological and
microanatomical details. However, it is important to remember that this method may
irreversibly modify epitopes recognized by antibodies. This can lead to considerable
differences in the reactivity of formalin fixed as compared to frozen tissue sections.
Many antibodies react differently and show a loss of reactivity between the two fixation
methods, or an altered staining pattern. A few facts may help to understand how to deal
with formalin, tissue fixation and immunohistochemistry on formalin-fixed, paraffin
embedded tissue sections.

Formaldehyde and Formalin

Formaldehyde is a pungent smelling gas that readily dissolves in water up to a
concentration of 37% - 40%. The aqueous solution of formaldehyde is called formalin.
Formaldehyde is chemically reactive, and is toxic. Avoid breathing fumes and protect
skin and eyes.

Formation of Polymers
Dissolved formaldehyde reacts with water to form methylene glycol, with the equilibrium
shifted to the methylene glycol side:

Formaldehyde Methylene Glycol

Methylene glycol tends to polymerize into long chains and thereby form an insoluble
white precipitate. This precipitate which can often be seen as a white layer on the
bottom is called paraformaldehyde. Note that polymerization reduces the number of
reactive formaldehyde molecules in solution and thus the power to fix the tissue to be
treated.

Sunlight increases polymerization. Therefore, formalin solutions should be

kept in a brown bottle in the dark. The addition of 10%-15% methanol helps to
prevent polymerization and formation of precipitate.

Cannizzaro Reaction and Buffered Formalin

Light and heat enable the Cannizzaro reaction, a disproportionation whereby two
molecules of formaldehyde form one molecule of formic acid and one of methanol:

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Cannizzaro reaction (R = H with formaldehyde)

The formic acid generated in this way decreases the pH and may negatively affect the
inegrity of an epitope of interest. Therefore, formalin solutions are generally buffered to
achieve optimal tissue fixation. A standard buffered formalin solution can be made up as
follows:

Buffered Formalin Solution:

Na2HPO4 6.5g (46mM final concentration)

NaH2PO4 H2O 4.0g (29mM final concentration)
Distilled water 900ml
37%-40% formalin 100ml

Check pH to be 7.4.

Buffering this solution and keeping it cool and dark increases its lifetime.

Formalin concentrations of 4% versus 10%

The fixation of tissues for immunohistochemical purposes is usually done with a 1 in 10
dilution of aqueous 37% - 40% formaldehyde. This yields a solution containing 3.7% -
4% formaldehyde.
Some protocols consider saturated formalin (37-40%) as „100%“. A 1 in 10 dilution of
this solution can be considered “10%”, but actually corresponds to a formaldehyde
concentration of 3.7% - 4%.

Mode of Action of Formalin

Formalin enables the cross-linking of proteins with little denaturing. Formaldehyde easily
reacts with many chemical groups, with amino groups of particular importance in the
context of tissue fixation. Free amino groups, such as the ones found with lysine, are
targeted preferentially, leading to altered epitope characteristics.
The duration of the fixation process varies with the nature of the tissue being treated,
and with its size. Any length of time from hours to weeks may be ideal and has to be
determined empirically.

Improving the reactivity of formalin-fixed tissues

With a little luck, a particular antibody will react comparably with frozen and formalin
fixed tissue sections. Sometimes formalin fixed sections may simply need a higher
antibody concentration to yield satisfactory results. However, formalin fixed tissue
sections often require a particular treatment in order to reverse the effect of formalin
fixation, a so-called epitope retrieval process. And many, if not most, epitopes are

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irreversibly lost through formalin fixation and require antibodies particularly developed
for this application.

Antigen / epitope retrieval techniques

The formalin-induced chemical modification of the epitope recognized by the antibody
may be reversed to a variable degree by an antigen (or epitope) retrieval treatment. For
proteolytic antigen retrieval, a variety of enzyme solutions such as Proteinase K,
pronase, Trypsin, or pepsin may yield optimal results. For heat-induced epitope retrieval
(HIER), a microwave oven or pressure cooker is used, where the tissue sections are
boiled in citrate or other appropriate buffer. Two standard methods routinely used at
BMA are antigen retrieval by heat treatment at low pH, and antigen retrieval by
enzymatic digestion with Proteinase K. Keep in mind that different epitope retrieval
procedures may yield different reaction patterns with some antibodies.

I. Heat-induced antigen retrieval using microwave oven and citrate buffer

Place slides in a heat resistant holder and put it into a cuvette with 0.01mM citrate
buffer, pH 6.0. Boil slides for 2x 7 minutes at 700 Watt in a microwave oven. Refill
evaporated buffer after 1x 7 minutes boiling. Cool cuvette to room temperature for 20
minutes (see www.bma.ch →protocols for a detailed protocol).
10mM Citrate buffer:
Dissolve 1.05g Citric acid monohydrate (MW 210.14) in 500ml distilled water, adjust pH
to 6.0 with 10M NaOH.

II. Proteolytic antigen retrieval using Proteinase K

Coat the entire section with Proteinase K diluted according to the manufacturer’s
instructions and incubate for approximately 7 minutes in a humid chamber at room
temperature. Incubation time may vary from 3 to 12 minutes depending on the antibody
and on the Proteinase supplier (see www.bma.ch →protocols for a detailed protocol).
Proteinase K (BMA Biomedicals, 20mg/ml. Product number T-3401):
Product T-3401 from BMA requires a 1 in 50 dilution for optimal reactivity. Dilution buffer
is 20mM Tris-HCl, pH 8.0.
20mM Tris-HCl, pH 8.0:
Dissolve 121mg Tris (MW 121) in 50ml distilled water, adjust pH to 8.0 with 1M HCl.
Combination of heat-induced and proteolytic antigen retrieval
In certain cases a combination of heat-induced and proteolytic antigen retrieval may be
beneficial.
A number of factors affect antibody binding in immunohistochemistry. Optimal
conditions should be evaluated and optimized for each individual antibody. Every
antibody produced by BMA has been validated for its use in immunohistochemistry
and data sheets of BMA products indicate if an antibody is suitable for use with
formalin-fixed tissues, and which one out of three standard procedures (citrate /
proteinase K / no treatment) yields satisfactory results.

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